Mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the mitochondrial processing peptidase

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mitochondrial presequences with a cleavage site that was confirmed by experimental studies can be grouped into three classes. Two major classes contain an arginine residue at position -2 or -3, and the third class does not have any conserved arginines. Sequence logos revealed loosely conserved cleavage motifs for the first two classes but no significant amino acid conservation for the third class. Investigation of processing determinants for a class III precursor, Nicotiana plumbaginifolia F1beta precursor of ATP synthase (pF1beta), was performed using a series of pF1beta presequence mutants and mutant presequence peptides derived from the C-terminal portion of the presequence. Replacement of -2 Gln by Arg inhibited processing, whereas replacement of either the most proximally located -5 Arg or -15 Arg by Leu had only a low inhibitory effect. The C-terminal portion of the pF1beta presequence forms a helix-turn-helix structure. Mutations disturbing or prolonging the helical element upstream of the cleavage site inhibited processing significantly. Structural models of potato MPP and the C-terminal pF1beta presequence peptide were built by homology modelling and empirical conformational energy search methods, respectively. Molecular docking of the pF1beta presequence peptide to the MPP model suggested binding of the peptide to the negatively charged binding cleft formed by the alpha-MPP and beta-MPP subunits in close proximity to the H111XXE114H115X(116-190)E191 proteolytic active site on beta-MPP. Our results show for the first time that the amino acid at the -2 position, even if not an arginine, as well as structural properties of the C-terminal portion of the presequence are important determinants for the processing of a class III precursor by MPP.     

Comparison of the Morphology Development of Polymer–Fullerene and Polymer–Polymer Solar Cells during Solution‐Shearing Blade Coating

In this work, the detailed morphology studies of polymer poly(3‐hexylthiophene‐2,5‐diyl) (P3HT):fullerene(PCBM) and polymer(P3HT):polymer naphthalene diimide thiophene (PNDIT) solar cell are presented to understand the challenge for getting high performance all‐polymer solar cells. The in situ X‐ray scattering and optical interferometry and ex situ hard and soft X‐ray scattering and imaging techniques are used to characterize the bulk heterojunction (BHJ) ink during drying and in dried state. The crystallization of P3HT polymers in P3HT:PCBM bulk heterojunction shows very different behavior compared to that of P3HT:PNDIT BHJ due to different mobilities of P3HT in the donor:acceptor glass. Supplemented by the ex situ grazing incidence X‐ray diffraction and soft X‐ray scattering, PNDIT has a lower tendency to form a mixed phase with P3HT than PCBM, which may be the key to inhibit the donor polymer crystallization process, thus creating preferred small phase separation between the donor and acceptor polymer.     

Influence of micronization (fine grinding) of soya bean meal and fullfat soya bean on productive performance and digestive traits in young pigs

Two trials were conducted to test the effect of micronization (very fine grinding) of soya bean meal (SBM) and fullfat soya bean (FFSB) on productive performance and digestive traits of piglets. The experimental design was completely randomized with four treatments arranged factorially (SBM and FFSB, micronized and ground). The mean particle size (MPS) was 47 and 881 μm for the SBM and 41 and 778 μm for the FFSB, micronized and ground, respectively. In trial 1 growth traits from 23 to 45 days of age and the coefficient of total tract apparent digestibility (CTTAD) of dietary components at 33 and 45 days of age were assessed. In trial 2 the coefficient of ileal apparent digestibility (CIAD) of dietary components, the pH of the gastro intestinal tract (GIT) and the weight of digestive organs and spleen were measured at 45 days of age. From 23 to 33 days of age pigs fed SBM grew faster (253 g/d versus 213 g/day; P<0.05) and were more efficient (0.87 g/g versus 0.98 g/g; P<0.01) than pigs fed FFSB. For the entire experiment (23–45 days of age) pigs fed SBM tended to grow more (360 g/day versus 324 g/day) and to eat more feed (414 g/day versus 380 g/day) than pigs fed FFSB (P<0.10). The CTTAD of crude protein (0.798 g/kg versus 0.778 g/kg), organic matter (0.864 g/kg versus 0.839 g/kg) and gross energy (0.849 g/kg versus 0.830 g/kg) were higher for pigs fed SBM than for pigs fed FFSB (P<0.001). In addition, CIAD of organic matter (0.765 g/kg versus 0.705 g/kg) and gross energy (0.761 g/kg versus 0.711 g/kg) were higher for SBM than for FFSB diets (P<0.001). The pH of the different segments of the GIT was not affected by the protein source (P>0.10). Particle size did not affect any trait studied (P>0.10). The poor performance and digestibility of pigs fed FFSB as compared to pigs fed SBM might be related to the conditions applied during processing. It is concluded that pigs fed soya bean meal perform better than pigs fed FFSB and that micronization of the soya protein sources does not affect any trait studied.     

欧洲的栗子业

欧洲的栗树栽培和利用历史悠久,其特产的欧洲栗,是当地重要的干果和硬木一源。意大利是欧洲栗子生产和加工的主要国家。西文概述了欧洲栗的分布、生产和科研,指出由于墨水 病和栗疫病的为害导臻欧洲栗产量急剧下降,有一些地区种质流失严重。     

Biosynthesis and transport of envelope proteins of Escherichia coli

Bacterial protein synthesis takes place in the cytoplasm, thus periplasmic and outer membrane proteins pass through the cytoplasmic membrane during their dispatch to the cell envelope. The exported proteins are synthesized as precursor that contains an extra amino-terminal sequence of amino-acids. This sequence, termed "signal sequence", is essential for transport of the envelope proteins through the inner membrane and is cleaved during the exportation process. Various hypotheses for the mechanism have been presented, and it is likely that no signal model will be suitable to the export of all cell envelope proteins. This review is focused on the relationship between the cytoplasmic membrane and the precursor form. The physiological state of the membrane - fluidity, membrane potential for instance - is the strategic requirement of exportation process. Precursors can be accumulated in whole cells with various treatments which alter the cytoplasmic membrane. This inhibition of processing is obtained by modification of unsaturated to saturated fatty acids ratio or with phenylethyl alcohol which perturbs the membrane fluidity, with uncoupler agents such as carbonyl cyanide m-chlorophenyl hydrazone which dissipate the proton motive force, or with hybrid proteins which get jamming in the membrane. However, little is known about the early steps of translocation process across the cytoplasmic membrane ; for instance, it is not clear yet whether energy is required for either or both of the first interaction membrane-precursor and the crossing through the membrane. Several studies have recently shown the presence of exportation sites and of proteins which might play a prominent role in the export process, but the mechanism of discrimination between outer membrane proteins and periplasmic proteins is unknown. Considerable work has been done by genetic or biochemical methods and we have now the first lights of the expert mechanism.     

Building protein interaction maps for Down's syndrome.

Now that the complete sequences for human chromosome 21 and the orthologous mouse genomic regions are known, reasonably complete, conserved, protein-coding gene catalogues are also available. The central issue now facing Down's syndrome researchers is the correlation of increased expression of specific, normal, chromosome 21 genes with the development of specific deficits in learning and memory. Because of the number of candidate genes involved, the number of alternative splice variants of individual genes and the number of pathways in which these genes function, a pathway analysis approach will be critical to success. Here, three examples, both gene specific and pathway related, that would benefit from pathway analysis are discussed: (1) the potential roles of eight chromosome 21 proteins in RNA processing pathways; (2) the chromosome 21 protein intersectin 1 and its domain composition, alternative splicing, protein interactions and functions; and (3) the interactions of ten chromosome 21 proteins with components of the mitogen-activated protein kinase and the calcineurin signalling pathways. A productive approach to developing gene-phenotype correlations in Down's syndrome will make use of known and predicted functions and interactions of chromosome 21 genes to predict pathways that may be perturbed by their increased levels of expression. Investigations may then be targeted in animal models to specific interactions, intermediate steps or end-points of such pathways and the downstream - perhaps amplified - consequences of gene dosage directly assessed. Once pathway perturbations have been identified, the potential for rational design of therapeutics becomes practical.     

Mitochondrial import of Omi: the definitive role of the putative transmembrane region and multiple processing sites in the amino-terminal segment

The mitochondrial serine protease Omi/HtrA2 has a proapoptotic role in mammalian cells. However, neither the topology nor the processing of Omi in mitochondria is clearly understood. To determine the topology of Omi in the mitochondrial IMS, EGFP fusions were expressed with the entire N-terminal segment of full-length Omi (FL-Omi) (133-EGFP), and that without the transmembrane region (DeltaTM-EGFP) in the cells. Immunocytochemical staining and alkaline extraction experiments revealed that the TM determines the topology of Omi in the IMS and anchors the pro form into the inner membrane. As a result, the protease and the PDZ domains are exposed to the IMS. Mature Omi largely exists in the IMS as a soluble form. The processing sites of the precursor protein were examined by in vitro import experiments. The import of the processing mutants revealed importance of Arg80, Arg91, and Arg93 residues for the processing of the N-terminal segment of FL-Omi. These results suggest that the N-terminal segment of FL-Omi contains multiple processing sites processed by matrix processing proteases.     

Production of bioactive peptides in an in vitro system

An in vitro system for the preparation of bioactive peptides is described. This system couples three different posttranslational modification enzymes, prohormone convertases (PCs), carboxypeptidase E, and peptidyl alpha-amidating enzyme, to transform recombinant precursors into bioactive peptides. Three different precursors, mouse proopiomelanocortin (mPOMC), rat proenkephalin (rPE), and human proghrelin, were used as model systems. The conversion of mPOMC and rPE to smaller peptide products was measured by radioimmunoassay. After optimization of the system, excellent efficiency was obtained: about 85% of starting mPOMC was converted to des-acetyl alpha-melanocyte-stimulating hormone (alpha-MSH). For proenkephalin, 75 and 96% yields were obtained for the opioid peptides Met-RGL and Met-enk, respectively. Cell-based assays demonstrated that in-vitro-generated des-acetyl alpha-MSH successfully activated the melanocortin 4 receptor. Proghrelin digestion was used to screen the specificity of PC cleavage and to confirm the cleavage site by mass spectroscopy. Mature ghrelin was produced by human furin, mouse prohormone convertase 1, and human prohormone convertase 7 but not by mouse prohormone convertase 2. These results demonstrate that our in vitro system (1) can produce peptides in quantities sufficient to carry out functional analyses, (2) can be used to determine the specificity of proprotein convertases on recombinant precursors, and (3) has the potential to identify novel peptide functions on both known and orphan G-protein-coupled receptors.     

Purification of chondroitin precursor from Escherichia coli K4 fermentation broth using membrane processing

Recently the possibility of producing the capsular polysaccharide K4, a fructosylated chondroitin, in fed-batch experiments was assessed. In the present study, a novel downstream process to obtain chondroitin from Escherichia coli K4 fermentation broth was developed. The process is simple, scalable and economical. In particular, downstream procedures were optimized with a particular aim of purifying a product suitable for further chemical modifications, in an attempt to develop a biotechnological platform for chondroitin sulfate production. During process development, membrane devices (ultrafiltration/diafiltration) were exploited, selecting the right cassette cut-offs for different phases of purification. The operational conditions (cross-flow rate and transmembrane pressure) used for the process were determined on an ?KTA cross-flow instrument (GE Healthcare, USA), a lab-scale automatic tangential flow filtration system. In addition, parameters such as selectivity and throughput were calculated based on the analytical quantification of K4 and defructosylated K4, as well as the major contaminants. The complete downstream procedure yielded about 75% chondroitin with a purity higher than 90%.